Date

2022-10-31

Author

Danışık, Nurseda

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The development of drug resistance in tumor cells is one of the biggest problems currently in the clinic. As the pace of drug discovery slows and each new drug becomes increasingly expensive to bring to market, it is becoming clearer that better model systems and an understanding of drug resistance mechanisms are needed for second-line therapies. The overall aim of this project is to investigate the clonal evolution and drug resistance in colorectal cancer cell lines Caco-2 and HT-29 by using cellular barcoding technology. It was hypothesized that the detection and tracking of the capecitabine-resistant clones in barcoded Caco-2 and irinotecan-resistant clones in barcoded HT-29 cell lines will unravel previously unidentified but key mechanisms. In parallel, studying the secondary drug responses and synergistic effects of drugs in resistant cell lines will help to identify new drug candidates and new treatment strategies. For these purposes, firstly Caco-2 and HT-29 cells were barcoded with unique barcode sequences by using a lentiviral vector system. Barcoded Caco-2 and HT-29 cells were treated with capecitabine and irinotecan respectively for six months to establish their drug-resistant derivatives. After achieving capecitabine-resistant Caco-2 and irinotecan-resistant HT-29 cells, DNA barcode sequencing and bioinformatics analysis were performed. According to DNA barcode analysis, capecitabine-resistant Caco-2, and irinotecan-resistant HT-29 clones were detected and time-dependent frequencies of these resistant clones in the populations were tracked by sequencing of floating dead barcoded Caco-2 and HT-29 cells which were collected as pellets from harvested mediums in equal intervals during drug treatment. Additionally, secondary-drug response curves showed that the capecitabine-resistant Caco-2 cells were more sensitive to SN-38, cetuximab, and oxaliplatin whereas the irinotecan-resistant HT-29 cells were more sensitive to dabrafenib. The results indicate that well-design model systems are crucial to understanding and finding new feasible treatment strategies for drug-resistance problems in the clinic, additionally, further use of cellular barcoding technology in in vivo studies can play an important role to solve the clonal evolution mechanism of drug resistance.

Subject Keywords

Colorectal cancer, Drug resistance, Barcoding technology

URI

https://hdl.handle.net/11511/99799

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Graduate School of Natural and Applied Sciences, Thesis